Serotonin (5HT) plays a crucial role in aggression, cognition, eating, mood, motor activity, pain, and sleep. 5HT transporters (SERTs) are neuronal membrane proteins that regulate, via uptake, extracellular 5HT concentrations. SERTs are also of interest because they are targets of therapeutics (e.g., antidepressants) and drugs of abuse (e.g., cocaine, amphetamine). Interestingly, SERTs have multiple conducting states, sometimes functioning as ion channels, and other times functioning more as traditional transporters of 5HT. However, the regulatory factors that determine which state SERT occupies, and the functional roles of these conducting states in neurons that endogenously express SERT are unknown. Preliminary data show that the conducting state that SERT occupies depends on SERT's interaction with other proteins. Aim 1 tests the hypothesis that calcium shifts SERT between its states by influencing this interaction, thus providing a physiological mechanism for regulating SERT function. Aim 2 examines the signal transduction pathways by which calcium influences which state SERT occupies. Aim 3 examines state-dependent differences in 5HT uptake. Aim 4 tests the hypothesis that amphetamine dysregulates the calcium-mediated shift in SERT conductance states, and examines the mechanisms underlying this effect. Aim 5 examines the role that these states play in thalamocortical neurons that endogenously express SERT by examining state-dependent cell excitability in the presence of SERT substrates. The experiments will be performed using biochemical, pharmacological, and electrophysiological approaches in cell expression systems and in neurons that endogenously express SERT. These experiments will add to our understanding of normal SERT function and how drugs of abuse that target SERT may mediate their effects. Understanding the factors that regulate SERT may also be important for the design of strategies useful in the treatment of serotonin transporter-mediated disorders.